Over the years, microbiologists have developed various methods for the storage or preservation of microorganisms, including subculturing, drying, freezing-drying, and freezing. Other methods, such as storage under liquid paraffin, in distilled water, and liquid drying (i.e., L-drying), have also been used with mixed success (See, e.g., K. A. Malik, "Maintenance of Microorganisms by Simple Methods," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 121-132). However, no one method fulfills all of the needs of each culture; the choice of method is often the result of weighing the advantages and disadvantages of multiple methods (See e.g., J. J. S. Snell, "General Introduction to Maintenance Methods," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 21-30).
Subculturing
Subculturing involves the periodic transfer of organisms to fresh growth media. This new culture (i.e., subculture) is allowed to grow under the appropriate conditions, its growth is slowed by refrigeration or other means, and it is used to replace the old culture. The time that is allowed to elapse between production of each subculture primarily depends upon the organism to be preserved. For example, subcultures of some organisms will remain viable for several years (e.g., some Staphylococcus cultures), while others require subculturing after only a few weeks or days (e.g., Neisseria). Although subculturing is attractive in some respects, there are many drawbacks to the method. For example, contamination presents a major problem that is present at each subculturing step. Contamination presents significant problems, as for most uses of preserved cultures, it is highly desired that the culture be pure. If a preserved culture becomes contaminated, there is the risk that the contaminants will outgrow and/or kill the original culture. Furthermore, even if there are viable organisms of interest in the contaminated culture, it may be very difficult to separate them from the contaminants. Thus, a contaminated culture is of questionable value, and it is critical to constantly monitor the purity of subcultures during the subculturing process, for the life of the culture.
In addition to the dangers of contamination, loss of viability is a constant concern. Problems with the preservation media (e.g., use of an inappropriate medium for the culture to be preserved), and dehydration of the cultures must be avoided. Storage of organisms in water, layering oil on top of the grown culture, use of media with limited nutrients and minimal carbohydrates, and for most organisms, lowering the storage temperature, may be helpful.
Furthermore, cultures preserved by continuous subculturing are very prone to genetic changes and selection. This risk increases with each subculture. Use of large inocula may help to reduce the risk, but it may increase the risk of contamination. In sum, subculturing is often convenient in situations where it is often necessary to use the culture, as it avoids the necessity of continually freezing and thawing frozen cultures or utilizing the entire lyophilized culture, if the culture has been freeze-dried. However, it is important that the culture be checked for purity before its use.
Drying
Desiccation has been widely used as a method to preserve microorganisms. A variety of methods are used, although all depend upon the removal of water from the culture and prevention of rehydration. Although drying methods have been more commonly used with molds than bacteria, some bacteria and yeasts have been successfully preserved using these methods. In the most commonly used methods, the cultures are dried in soil, sand, kieselguhr, and silica gel, dried onto paper or gelatin strips or discs, or pre-dried plugs.
None of the drying methods has found universal acceptance, as their efficacy appears to be culture-specific (i.e., some organisms may not be dried as they become non-viable during the process). For cultures that are suited for preservation by drying, long-term viability is often good, contamination is less likely than with subculturing, and capital equipment costs are small. However, stability of strain characteristics appears to be strain-specific and genetic changes have been reported.
Freezing
In freezing, water is made unavailable to the organisms, and the dehydrated cells are maintained at low temperatures. Damage may be caused to the cells during the cooling stage and/or the subsequent thawing. This damage may be caused either by the concentration of electrolytes through removal of water as ice, or by the formation of ice crystals that shear the cells. Damage may be somewhat limited by adjusting the cooling and warming rates, as well as by adding cryoprotectants (e.g., dimethyl sulfoxide (DMSO), glycerol, or blood) to the cell suspension. Although various temperatures have been used to store frozen cultures (e.g., -20.degree., -30.degree., -40.degree., -70.degree., -140.degree., and -196.degree. C.), poor results are usually observed at temperatures above -30.degree. C.
Freezing in liquid nitrogen has been widely used for many organisms (e.g., bacteria, fungi, protozoa, etc.), and is currently recommended for storage of valuable seed stock cultures. There are numerous advantages to this method, as virtually no loss of viability occurs during storage (although some cells may die during the cooling and warming); in general, there is no genetic change or loss of characters; and the longevity and stability is greater for most cultures than that obtainable by freeze-drying (See, E. R. James, "Maintenance of Parasitic Protozoa by Cryopreservation," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 209-226; P. H. Calcott and A. M. Gargett, FEMS Microbiol. Lett., 10:151-155 1981!; D. L. Williams and P. H. Calcott, J. Gen. Microbiol., 128:215-218 1982!; and B. E. Kirsop, "Maintenance of Yeasts," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 161-182).
Disadvantages of freezing cultures in liquid nitrogen include the need to continually replenish the liquid nitrogen, the high cost of equipment, the risk of explosion if glass containers are used, storage space may become problematic, and the method is not very convenient for distribution of large numbers of cultures.
A relatively recently described method for freezing and storing cultures on beads has been successful (See, R. K. A. Feltham et al., J. Appl. Bacteriol., 44:313-316 1978!; and Jones et al., "Maintenance of Bacteria on Glass Beads at -60.degree. C. to -76.degree. C.," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 45-50). This method is very quick, easy to perform, requires minimal storage space, and requires no manipulation during storage. The main disadvantages are related to the costs of the freezer, as well as the equipment necessary to monitor and maintain the low temperature of the cultures.
In addition, methods have been developed to preserve various microorganisms by deep freezing and cryopreservation, in which the cells are pre-dried onto desiccated carriers such as silica gel, glass beads, polymeric materials, filter paper strips, other materials (See, T. M. Sidyakina, "Low Temperature Freezing of Microorganisms on Silica Gel," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 65-70; and K. A. Malik, "Maintenance of Microorganisms by Simple Methods," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 121-132). Cells that have been immobilized have been reported to retain their viability and characteristics much better than free cells (See, Sidyakina supra). However, these methods require additional manipulation during the process, and may be too cumbersome for some situations.
Freeze-Drying (Lyophilization)
Freeze-drying involves the removal of water by sublimination from a frozen culture. Organisms are grown on a suitable growth medium, aliquots are suspended in an appropriate freeze-drying liquid in ampules or vials, and placed in the freeze-drying apparatus, where they are frozen, and exposed to a vacuum. The water vapor from the culture is either trapped in a refrigerated condenser unit, or in phosphorous pentoxide. After freeze-drying, the cultures are sealed in their vials, often under vacuum or in an inert gas, and are stored at room temperature, refrigerated, or frozen. Two methods of freeze-drying are commonly used in industry, namely centrifugal and shelf freeze-drying (See, R. H. Rudge, "Maintenance of Bacteria by Freeze-Drying," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, 1991!, pp. 31-44).
Although freeze drying has been widely used to preserve various organisms, there are problems associated with this method. For example, glass ampules are generally sealed closed with a flame (e.g., a torch), requiring some care in order to avoid injury to the operator, and some ampules are very difficult to open, requiring filing in order to sufficiently weaken the glass so that the ampule can be broken. This presents risks of contamination of the culture through the introduction of contaminants through the filed area of the ampule, as well as risk of injury to the operator, should the ampule unexpectedly break. In addition, there is the risk of injury and inoculation from the broken glass (i.e., the operator may be cut on the edge of the broken glass and be inoculated with the organisms present in the ampule). Thus, there are major safety considerations associated with the use of freeze drying methods.
Despite the number of methods available for preservation of microorganisms, it is clear that improved methods are needed. Improved methods and devices should be economical, easy and safe to use and transport, and provide for long-term viability of preserved cultures.